Inbred corn line 7RN401

ABSTRACT

An inbred corn line, designated 7RN401, is disclosed. The invention relates to the seeds of inbred corn line 7RN401, to the plants of inbred corn line 7RN401 and to methods for producing a corn plant, either inbred or hybrid, by crossing the inbred line 7RN401 with itself or another corn line. The invention further relates to methods for producing a corn plant containing in its genetic material one or more transgenes and to the transgenic plants produced by that method and to methods for producing other inbred corn lines derived from the inbred 7RN401.

BACKGROUND OF THE INVENTION

The present invention relates to a new and distinctive corn inbred line,designated 7RN401. There are numerous steps in the development of anynovel, desirable plant germplasm. Plant breeding begins with theanalysis and definition of problems and weaknesses of the currentgermplasm, the establishment of program goals, and the definition ofspecific breeding objectives. The next step is selection of germplasmthat possess the traits to meet the program goals. The goal is tocombine in a single variety or hybrid an improved combination ofdesirable traits from the parental germplasm. These important traits mayinclude higher yield, resistance to diseases and insects, better stalksand roots, tolerance to drought and heat, and better agronomic quality.

Choice of breeding or selection methods depends on the mode of plantreproduction, the heritability of the trait(s) being improved, and thetype of cultivar used commercially (e.g., F₁ hybrid cultivar, purelinecultivar, etc.). For highly heritable traits, a choice of superiorindividual plants evaluated at a single location will be effective,whereas for traits with low heritability, selection should be based onmean values obtained from replicated evaluations of families of relatedplants. Popular selection methods commonly include pedigree selection,modified pedigree selection, mass selection, and recurrent selection.

The complexity of inheritance influences choice of the breeding method.Backcross breeding is used to transfer one or a few favorable genes fora highly heritable trait into a desirable cultivar. This approach hasbeen used extensively for breeding disease-resistant cultivars. Variousrecurrent selection techniques are used to improve quantitativelyinherited traits controlled by numerous genes. The use of recurrentselection in self-pollinating crops depends on the ease of pollination,the frequency of successful hybrids from each pollination, and thenumber of hybrid offspring from each successful cross.

Each breeding program should include a periodic, objective evaluation ofthe efficiency of the breeding procedure. Evaluation criteria varydepending on the goal and objectives, but should include gain fromselection per year based on comparisons to an appropriate standard,overall value of the advanced breeding lines, and number of successfulcultivars produced per unit of input (e.g., per year, per dollarexpended, etc.).

Promising advanced breeding lines are thoroughly tested and compared toappropriate standards in environments representative of the commercialtarget area(s) for three years at least. The best lines are candidatesfor new commercial cultivars; those still deficient in a few traits areused as parents to produce new populations for further selection.

These processes, which lead to the final step of marketing anddistribution, usually take from eight to 12 years from the time thefirst cross is made. There fore, development of new cultivars is atime-consuming process that requires precise forward planning, efficientuse of resources, and a minimum of changes in direction.

A most difficult task is the identification of individuals that aregenetically superior, because for most traits the true genotypic valueis masked by other confounding plant traits or environmental factors.One method of identifying a superior plant is to observe its performancerelative to other experimental plants and to a widely grown standardcultivar. If a single observation is inconclusive, replicatedobservations provide a better estimate of its genetic worth.

The goal of plant breeding is to develop new, unique and superior corninbred lines and hybrids. The breeder initially selects and crosses twoor more parental lines, followed by repeated selfing and selection,producing many new genetic combinations. The breeder can theoreticallygenerate billions of different genetic combinations via crossing,selfing and mutations. The breeder has no direct control at the cellularlevel. Therefore, two breeders will never develop the same line, or evenvery similar lines, having the same corn traits.

Each year, the plant breeder selects the germplasm to advance to thenext generation. This germplasm is grown under unique and differentgeographical, climatic and soil conditions, and further selections arethen made, during and at the end of the growing season. The inbred lineswhich are developed are unpredictable. This unpredictability is becausethe breeder's selection occurs in unique environments, with no controlat the DNA level (using conventional breeding procedures), and withmillions of different possible genetic combinations being generated. Abreeder of ordinary skill in the art cannot predict the final resultinglines he develops, except possibly in a very gross and general fashion.The same breeder cannot produce the same line twice by using the exactsame original parents and the same selection techniques. Thisunpredictability results in the expenditure of large research monies todevelop a superior new corn inbred line.

The development of commercial corn hybrids requires the development ofhomozygous inbred lines, the crossing of these lines, and the evaluationof the crosses. Pedigree breeding and recurrent selection breedingmethods are used to develop inbred lines from breeding populations.Breeding programs combine desirable traits from two or more inbred linesor various broad-based sources into breeding pools from which inbredlines are developed by selfing and selection of desired phenotypes. Thenew inbreds are crossed with other inbred lines and the hybrids fromthese crosses are evaluated to determine which have commercialpotential.

Pedigree breeding is used commonly for the improvement ofself-pollinating crops or inbred lines of cross-pollinating crops. Twoparents which possess favorable, complementary traits are crossed toproduce an F₁. An F₂ population is produced by selfing one or severalF₁'s or by intercrossing two F₁'s (sib mating). Selection of the bestindividuals is usually begun in the F₂ population; then, beginning inthe F₃, the best individuals in the best families are selected.Replicated testing of families, or hybrid combinations involvingindividuals of these families, often follows in the F₄ generation toimprove the effectiveness of selection for traits with low heritability.At an advanced stage of inbreeding (i.e., F₆ and F₇), the best lines ormixtures of phenotypically similar lines are tested for potentialrelease as new cultivars.

Mass and recurrent selections can be used to improve populations ofeither self-or cross-pollinating crops. A genetically variablepopulation of hetero-zygous individuals is either identified or createdby intercrossing several different parents. The best plants are selectedbased on individual superiority, outstanding progeny, or excellentcombining ability. The selected plants are intercrossed to produce a newpopulation in which further cycles of selection are continued.

Backcross breeding has been used to transfer genes for a simplyinherited, highly heritable trait into a desirable homozygous cultivaror inbred line which is the recurrent parent. The source of the trait tobe transferred is called the donor parent. The resulting plant isexpected to have the attributes of the recurrent parent (e.g., cultivar)and the desirable trait transferred from the donor parent. After theinitial cross, individuals possessing the phenotype of the donor parentare selected and repeatedly crossed (backcrossed) to the recurrentparent. The resulting plant is expected to have the attributes of therecurrent parent (e.g., cultivar) and the desirable trait transferredfrom the donor parent.

Descriptions of other breeding methods that are commonly used fordifferent traits and crops can be found in one of several referencebooks (e.g., Allard, 1960; Simmonds, 1979; Sneep et al., 1979; Fehr,1987).

Proper testing should detect any major faults and establish the level ofsuperiority or improvement over current cultivars. In addition toshowing superior performance, there must be a demand for a new cultivarthat is compatible with industry standards or which creates a newmarket. The introduction of a new cultivar will incur additional coststo the seed producer, the grower, processor and consumer; for specialadvertising and marketing, altered seed and commercial productionpractices, and new product utilization. The testing preceding release ofa new cultivar should take into consideration research and developmentcosts as well as technical superiority of the final cultivar. Forseed-propagated cultivars, it must be feasible to produce seed easilyand economically.

Once the inbreds that give the best hybrid performance have beenidentified, the hybrid seed can be reproduced indefinitely as long asthe homogeneity of the inbred parent is maintained. A single-crosshybrid is produced when two inbred lines are crossed to produce the F₁progeny. A double-cross hybrid is produced from four inbred linescrossed in pairs (A×B and C×D) and then the two F₁ hybrids are crossedagain (A×B)×(C×D). Much of the hybrid vigor exhibited by F₁ hybrids islost in the next generation (F₂). Consequently, seed from hybridvarieties is not used for planting stock.

Hybrid corn seed is typically produced by a male sterility systemincorporating manual or mechanical detasseling. Alternate strips of twocorn inbreds are planted in a field, and the pollen-bearing tassels areremoved from one of the inbreds (female). Providing that there issufficient isolation from sources of foreign corn pollen, the ears ofthe detasseled inbred will be fertilized only from the other inbred(male), and the resulting seed is therefore hybrid and will form hybridplants.

The laborious, and occasionally unreliable, detasseling process can beavoided by using cytoplasmic male-sterile (CMS) inbreds. Plants of a CMSinbred are male sterile as a result of factors resulting from thecytoplasmic, as opposed to the nuclear, genome. Thus, thischaracteristic is inherited exclusively through the female parent incorn plants, since only the female provides cytoplasm to the fertilizedseed. CMS plants are fertilized with pollen from another inbred that isnot male-sterile. Pollen from the second inbred may or may notcontribute genes that make the hybrid plants male-fertile. Seed fromdetasseled fertile corn and CMS produced seed of the same hybrid can beblended to insure that adequate pollen loads are available forfertilization when the hybrid plants are grown.

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar et al. and chromosomal translocations asdescribed by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Theseand all patents referred to are incorporated by reference. In additionto these methods, Albertsen et al., U.S. Pat. No. 5,432,068 havedeveloped a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility, silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing, or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

There are many other methods of conferring genetic male sterility in theart, each with its own benefits and drawbacks. These methods use avariety of approaches such as delivering into the plant a gene encodinga cytotoxic substance associated with a male tissue specific promoter oran anti-sense system in which a gene critical to fertility is identifiedand an antisense to that gene is inserted in the plant (see,Fabinjanski, et al. EPO 89/3010153.8 publication no. 329, 308 and PCTapplication PCT/CA90/00037 published as WO 90/08828).

Another version useful in controlling male sterility makes use ofgametocides. Gametocides are not a genetic system, but rather a topicalapplication of chemicals. These chemicals affect cells that are criticalto male fertility. The application of these chemicals affects fertilityin the plants only for the growing season in which the gametocide isapplied (see Carlson, G.R., U.S. Pat. No. 4,936,904). Application of thegametocide, timing of the application and genotype specifically oftenlimit the usefulness of the approach.

Corn is an important and valuable field crop. Thus, a continuing goal ofplant breeders is to develop stable, high yielding corn hybrids that areagronom-ically sound. The reasons for this goal are obviously tomaximize the amount of grain produced on the land used and to supplyfood for both animals and humans. To accomplish this goal, the cornbreeder must select and develop corn plants that have the traits thatresult in superior parental lines for producing hybrids.

SUMMARY OF THE INVENTION

According to the invention, there is provided a novel inbred corn line,designated 7RN401. This invention thus relates to the seeds of inbredcorn line 7RN401, to the plants of inbred corn line 7RN401 and tomethods for producing a corn plant produced by crossing the inbred line7RN401 with itself or another corn line, and to methods for producing acorn plant containing in its genetic material one or more transgenes andto the transgenic corn plants produced by that method. This inventionalso relates to methods for producing other inbred corn lines derivedfrom inbred corn line 7RN401 and to the inbred corn lines derived by theuse of those methods. This invention further relates to hybrid cornseeds and plants produced by crossing the inbred line 7RN401 withanother corn line.

The inbred corn plant of the invention may further comprise, or have, acytoplasmic factor that is capable of conferring male sterility. Partsof the corn plant of the present invention are also provided, such ase.g., pollen obtained from an inbred plant and an ovule of the inbredplant.

In another aspect, the present invention provides regenerable cells foruse in tissue culture or inbred corn plant 7RN401. The tissue culturewill preferably be capable of regenerating plants having thephysiological and morphological characteristics of the foregoing inbredcorn plant, and of regenerating plants having substantially the samegenotype as the foregoing inbred corn plant. Preferably, the regenerablecells in such tissue cultures will be embryos, protoplasts, meristematiccells, callus, pollen, leaves, anthers, roots, root tips, silk, flowers,kernels, ears, cobs, husks or stalks. Still further, the presentinvention provides corn plants regenerated from the tissue cultures ofthe invention.

DEFINITIONS

In the description and tables which follow, a number of terms are used.In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided:

Predicted RM. This trait for a hybrid, predicted relative maturity (RM),is based on the harvest moisture of the grain. The relative maturityrating is based on a known set of checks and utilizes conventionalmaturity systems such as the Minnesota Relative Maturity Rating System.

MN RM. This represents the Minnesota Relative Maturity Rating (MN RM)for the hybrid and is based on the harvest moisture of the grainrelative to a standard set of checks of previously determined MN RMrating. Regression analysis is used to compute this rating.

Yield (Bushels/Acre). The yield in bushels/acre is the actual yield ofthe grain at harvest adjusted to 15% moisture.

Grain Moisture. The grain moisture is the actual percentage moisture ofthe grain at harvest as measured by the combine.

CTPS Index. The CTPS Index is calculated with values for yield,moisture, stalk lodging and root lodging, compared to the average of apredetermined set of official CTPS check hybrids.

Adjusted Test Weight. The Adjusted Test Weight is the weight in poundsper bushel which is adjusted for harvest grain moisture level.

GDU. The GDU (=heat unit) is a measure of the number of growing degreeunits (GDU) or heat units used in the tracking of flowering andmaturation of inbred lines and hybrids. Growing degree units arecalculated by the Barger Method, where the heat units for a 24-hourperiod are: ${GDU} = {\frac{( {{Max}.{+ {Min}}} )}{2} - 50.}$

The highest maximum used is 86° F. and the lowest minimum used is 50° F.For each hybrid, it takes a certain number of GDUs to reach variousstages of plant development. GDUs are a way of measuring plant maturity.

GDU Silk. The GDU Silk is the number of growing degree units afterplanting when 50% of the plants have extruded silk.

GDU Pollen. The GDU Pollen is the number of growing degree units afterplanting when 50% of the plants are shedding pollen.

Stalk Lodging. This is the percentage of plants that stalk lodge, i.e.,stalk breakage, as measured by either natural lodging or pushing thestalks determining the percentage of plants that break off below theear. This is a relative rating of a hybrid to other hybrids forstandability.

Root Lodging. The root lodging is the percentage of plants that rootlodge; i.e., those that lean from the vertical axis at an approximate30° angle or greater would be counted as root lodged. Included aregoose-necked plants previously counted as summer root lodged, but notincluded are plants root lodged due to damage caused by cultivators orridge-hill equipment.

Top Integrity. The Top Integrity is a rating of the condition of planttops late during the harvest season, based on the following scores:

9 =All top material intact, 100% to 91% leaves retained;

8=90-99% of top material intact, 90-75% leaves retained;

7=90-99% of top material intact, 74-0% leaves retained;

6=89-75% of top material intact;

5=74-50% of top material intact;

4=49-25% of top material intact;

3=24-10% of top material intact;

2=9-1% of top material intact; or

1=0% top material intact.

Plant Height. This is a measure of the height of the hybrid or inbredfrom the ground to the node of the flag leaf, and is measured in inchesor centimeters.

Ear Height. The ear height is a measure from the ground to the collar ofthe primary ear node, and is measured in inches or centimeters.

Dropped Ears. This is a measure of the number of plants per plot withears detached from the primary ear node. Does not include ears on theground that are attached to a section of stalk.

Emergence Vigor. The Emergence Vigor is an early visual rating of thehybrids emergence vigor. This is a 1-9 rating where 9 is the best vigor.

Early Vigor. The Early Vigor is a rating of the hybrids vigor when thestalks are between the researcher's calf and knee in height. This is a1-9 rating where 9 is the best vigor.

Count. Count refers to the total number of observations used in areported comparison.

Environment. Environment (env) refers to the number of locations wheretwo hybrids are grown together and in the same experiment.

Years. Years refers to the number of calendar years included in acomparison.

b. “b” is a regression value of hybrid yield and location (orenvironment) yield. The statistic is used as a measure of predictinghybrid responsiveness to higher yielding environments and is sometimesconsidered as a measure of stability.

Percent Oil. The Percent Oil is the measure of oil in the grain ofself-pollinated hybrid plants as measured by NIR (Near InfraredReflectance) or NIT (Near Infrared Transmittance).

Percent Protein. The Percent Protein is the measure percentage of crudeprotein in the grain of self-pollinated hybrid plants as measured by NIRor NIT.

Percent Starch. The Percent Starch is the measure of starch in the grainof self-pollinated hybrid plants as measured by NIR or NIT.

Disease Resistance. Ratings for the following diseases are shown fromreplicated inoculated disease screening trials. This is a 1-9 ratingwhere the higher number indicates a higher amount of resistance ortolerance to the disease. Examples of diseases include:

Gray Leaf Spot (Cercospora zeae-maydis)

Northern Corn Leaf Blight (Exserohilum turcicum)

Southern Corn Leaf Blight (Bipolaris maydis)

Eyespot (Kabatiella zeae)

Stewart's Wilt Leaf Blight (Erwinia stewarfii)

Fusarium Kernel Rot (Fusarium moniliforme)

ECB1 Average. The “ECB1 Average” is a rating from replicated screentrials infested with European Corn Borers (ECB) (Ostrinia nubilalis),where a higher rating indicates a higher amount of ECB damage. Allratings are for ECB1 (first generation European Corn Borer).

ECB1 Maximum. ECB1 Maximum reflects the highest rating recorded for ECB1across all environments.

Number of Observations (@ Obs). This refers to the number of ECB1ratings collected for the pair of hybrids in comparison.

Allele. The allele is any of one or more alternative forms of a gene,all of which alleles relate to one trait or characteristic. In a diploidcell or organism, the two alleles of a given gene occupy correspondingloci on a pair of homologous chromosomes.

Backcrossing. Backcrossing is a process in which a breeder repeatedlycrosses hybrid progeny back to one of the parents, for example, a firstgeneration hybrid F₁ with one of the parental genotypes of the F₁hybrid.

Essentially all the physiological and morphological characteristics. Aplant having essentially all the physiological and morphologicalcharacteristics means a plant having the physiological and morphologicalcharacteristics, except for the characteristics derived from theconverted gene.

Quantitative Trait Loci (QTL). Quantitative trait loci (QTL) refer togenetic loci that control to some degree numerically representabletraits that are usually continuously distributed.

Regeneration. Regeneration refers to the development of a plant fromtissue culture.

Single Gene Converted. Single gene converted or conversion plant refersto plants which are developed by a plant breeding technique calledbackcrossing wherein essentially all of the desired morphological andphysiological characteristics of an inbred are recovered in addition tothe single gene transferred into the inbred via the backcrossingtechnique or via genetic engineering.

DETAILED DESCRIPTION OF THE INVENTION

Inbred corn line 7RN401 is a yellow dent corn with superiorcharacteristics, and provides an excellent parental line in crosses forproducing first generation (F₁) hybrid corn.

Inbred corn line 7RN401 has the following morphologic and othercharacteristics (based primarily on morphological data collected at oneor more of the following locations: Marshalltown, Iowa; Huxley, Iowa;Arlington, Wis.; and Olivia, Minn. Disease ratings were collectedprimarily at Champaign, Ill. and other supporting stations. ECB ratingswere collected primarily in Huxley, Iowa. GDU data were collected inmulti-location flowering studies grown across the Midwest).

Inbred Description Information

1. TYPE: Dent

2. MATURITY:

Heat Units From emergence to 50% of plants in silk: 1625 From emergenceto 50% of plants in pollen 1643${{Heat}\quad {{Units}:}}\quad = {\frac{\begin{matrix}\lbrack {{{Max}.\quad {Temp}.\quad ( {\leq {86{^\circ}\quad {F.}}} )} +}  \\ {{Min}.\quad {Temp}.\quad ( {\geq {50{^\circ}\quad {F.}}} )} \rbrack\end{matrix}}{2} - 50}$

3. PLANT:

Plant Height (to tassel tip): 210 cm

Ear Height (to base of top ear): 80 cm

4. TASSEL:

Anther Color: Yellow—Munsell Code 5Y 8/6

Glume Color: Light green—Munsell Code 2.5GY 7/6

5. EAR: (Unhusked Data)

Silk Color (3 days after emergency): Pink—Munsell Code 2.5R 5/8

6. COB:

Cob Color: White

7. DISEASE RESISTANCE:

Stewart's Wilt Leaf Blight: Moderately Susceptable

Gray Leaf Spot: Moderately Susceptible

8. INSECT RESISTANCE:

European Corn Borer Race 1: Highly Susceptible

European Corn Borer Race 2: Susceptible

This invention is also directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plant,wherein the first or second corn plant is the inbred corn plant from theline 7RN401. Further, both first and second parent corn plants may befrom the inbred line 7RN401. Therefore, any methods using the inbredcorn line 7RN401 are part of this invention: selfing, backcrosses,hybrid breeding, and crosses to populations. Any plants produced usinginbred corn line 7RN401 as a parent are within the scope of thisinvention. Advantageously, the inbred corn line is used in crosses withother corn varieties to produce first generation (F₁) corn hybrid seedand plants with superior characteristics.

7RN401 is most similar to LH132, however there are numerous differencesincluding later maturity, higher yield per se and different silk and cobcolor. 7SH382 reaches 50% pollen shed at approximately 51 GDU's laterthan LH132 and 50% silk is approximately 45 GDU's later than LH132.Grain moisture of hybrids produced with 7RN401 are also slightly higherthan LH132 hybrids when crossed to the same testers. Ears of 7RN401 havepink silk and a white cob color, while LH132 ears have yellow-green silkand red cobs.

Some of the criteria used to select ears in various generations include:yield, stalk quality, root quality, disease tolerance, late plantgreenness, late season plant intactness, ear retention, pollen sheddingability, silking ability, and corn borer tolerance. During thedevelopment of the line, crosses were made to inbred testers for thepurpose of estimating the line's general and specific combining ability,and evaluations were run by the Research Station. The inbred wasevaluated further as a line and in numerous crosses by other ResearchStations across the Corn Belt. The inbred has proven to have a very goodcombining ability in hybrid combinations.

The inbred has shown uniformity and stability within the limits ofenvironmental influence for the traits. It has been self-pollinated andear-rowed a sufficient number of generations, with careful attention touniformity of plant type to ensure homozygosity and phenotypic stabilitynecessary to use in commercial production. The line has been increasedboth by hand and sibbed in isolated fields with continued observationsfor uniformity. No variant traits have been observed or are expected in7RN401.

Further Embodiments of the Invention

This invention also is directed to methods for producing a corn plant bycrossing a first parent corn plant with a second parent corn plantwherein either the first or second parent corn plant is an inbred cornplant of the line 7RN401. Further, both first and second parent cornplants can come from the inbred corn line 7RN401. Still further, thisinvention also is directed to methods for producing an inbred corn line7RN401-derived corn plant by crossing inbred corn line 7RN401 with asecond corn plant and growing the progeny seed, and repeating thecrossing and growing steps with the inbred corn line 7RN401-derivedplant from 0 to 7 times. Thus, any such methods using the inbred cornline 7RN401 are part of this invention: selfing, backcrosses, hybridproduction, crosses to populations, and the like. All plants producedusing inbred corn line 7RN401 as a parent are within the scope of thisinvention, including plants derived from inbred corn line 7RN401.Advantageously, the inbred corn line is used in crosses with other,different, corn inbreds to produce first generation (F₁) corn hybridseeds and plants with superior characteristics.

It should be understood that the inbred can, through routinemanipulation of cytoplasmic or other factors, be produced in amale-sterile form. Such embodiments are also contemplated within thescope of the present claims.

As used herein, the term plant includes plant cells, plant protoplasts,plant cell tissue cultures from which corn plants can be regenerated,plant calli, plant clumps and plant cells that are intact in plants orparts of plants, such as embryos, pollen, ovules, flowers, kernels,ears, cobs, leaves, husks, stalks, roots, root tips, anthers, silk andthe like.

Duncan, et al., Planta 165:322-332 (1985) reflects that 97% of theplants cultured that produced callus were capable of plant regeneration.Subsequent experiments with both inbreds and hybrids produced 91%regenerable callus that produced plants. In a further study in 1988,Songstad, et al., Plant Cell Reports 7:262-265 (1988), reports severalmedia additions that enhance regenerability of callus of two inbredlines. Other published reports also indicated that “nontraditional”tissues are capable of producing somatic embryogenesis and plantregeneration. K. P. Rao et al., Maize Genetics Cooperation Newsletter,60:64-65 (1986), refers to somatic embryogenesis from glume calluscultures and B. V. Conger, et al., Plant Cell Reports, 6:345-347 (1987)indicates somatic embryogenesis from the tissue cultures of corn leafsegments. Thus, it is clear from the literature that the state of theart is such that these methods of obtaining plants are, and were,“conventional” in the sense that they are routinely used and have a veryhigh rate of success.

Tissue culture of corn is described in European Patent Application,publication 160,390, incorporated herein by reference. Corn tissueculture procedures are also described in Green and Rhodes, “PlantRegeneration in Tissue Culture of Maize,” Maize for Biological Research(Plant Molecular Biology Association, Charlottesville, VA 367-372,(1982)) and in Duncan et al., “The Production of Callus Capable of PlantRegeneration from Immature Embryos of Numerous Zea Mays Genotypes,” 165Planta 322:332 (1985). Thus, another aspect of this invention is toprovide cells which upon growth and differentiation produce corn plantshaving the physiological and morphological characteristics of inbredcorn line 7RN401.

The utility of inbred corn line 7RN401 also extends to crosses withother species. Commonly, suitable species will be of the familyGraminaceae, and especially of the genera Zea, Tripsacum, Croix,Schlerachne, Polytoca, Chionachne, and Trilobachne, of the tribeMaydeae. Potentially suitable for crosses with 7RN401 may be the variousvarieties of grain sorghum, Sorghum bicolor (L.) Moench.

With the advent of molecular biological techniques that have allowed theisolation and characterization of genes that encode specific proteinproducts, scientists in the field of plant biology developed a stronginterest in engineering the genome of plants to contain and expressforeign genes, or additional, or modified versions of native, orendogenous, genes (perhaps driven by different promoters) in order toalter the traits of a plant in a specific manner. Such foreignadditional and/or modified genes are referred to herein collectively as“transgenes”. Over the last fifteen to twenty years several methods forproducing transgenic plants have been developed, and the presentinvention, in particular embodiments, also relates to transformedversions of the claimed inbred line.

Plant transformation involves the construction of an expression vectorwhich will function in plant cells. Such a vector comprises DNAcomprising a gene under control of or operatively linked to a regulatoryelement (for example, a promoter). The expression vector may contain oneor more such operably linked gene/regulatory element combinations. Thevector(s) may be in the form of a plasmid, and can be used alone or incombination with other plasmids, to provide transformed corn plants,using transformation methods as described below to incorporatetransgenes into the genetic material of the corn plant(s).

Expression Vectors for Corn Transformation

Marker Genes—Expression vectors include at least one genetic marker,operably linked to a regulatory element (a promoter, for example) thatallows transformed cells containing the marker to be either recovered bynegative selection, i.e., inhibiting growth of cells that do not containthe selectable marker gene, or by positive selection, i.e., screeningfor the product encoded by the genetic marker. Many commonly usedselectable marker genes for plant transformation are well known in thetransformation arts, and include, for example, genes that code forenzymes that metabolically detoxify a selective chemical agent which maybe an antibiotic or a herbicide, or genes that encode an altered targetwhich is insensitive to the inhibitor. A few positive selection methodsare also known in the art.

One commonly used selectable marker gene for plant transformation is theneomycin phosphotransferase ll (nptll) gene, isolated from transposonTn5, which when placed under the control of plant regulatory signalsconfers resistance to kanamycin. Fraley et al., Proc. Natl. Acad. Sci.U.S.A., 80:4803 (1983). Another commonly used selectable marker gene isthe hygromycin phosphotransferase gene which confers resistance to theantibiotic hygromycin. Vanden Elzen et al., Plant Mol. Biol., 5:299(1985).

Additional selectable marker genes of bacterial origin that conferresistance to antibiotics include gentamycin acetyl transferase,streptomycin phosphotransferase, aminoglycoside-3′-adenyl transferase,the bleomycin resistance determinant. Hayford et al., Plant Physiol.86:1216 (1988), Jones et al., Mol. Gen. Genet., 210:86 (1987), Svab etal., Plant Mol. Biol. 14:197 (1990<Hille et al., Plant Mol. Biol. 7:171(1986). Other selectable marker genes confer resistance to herbicidessuch as glyphosate, glufosinate or broxynil. Comai et al., Nature317:741-744 (1985), Gordon-Kamm et al., Plant Cell 2:603-618 (1990) andStalker et al., Science 242:419-423 (1988).

Other selectable marker genes for plant transformation are not ofbacterial origin. These genes include, for example, mouse dihydrofolatereductase, plant 5-enolpyruvylshikimate-3-phosphate synthase and plantacetolactate synthase. Eichholtz et al., Somatic Cell Mol. Genet. 13:67(1987), Shah et al., Science 233:478 (1986), Charest et al., Plant CellRep. 8:643 (1990).

Another class of marker genes for plant transformation require screeningof presumptively transformed plant cells rather than direct geneticselection of transformed cells for resistance to a toxic substance suchas an antibiotic. These genes are particularly useful to quantify orvisualize the spatial pattern of expression of a gene in specifictissues and are frequently referred to as reporter genes because theycan be fused to a gene or gene regulatory sequence for the investigationof gene expression. Commonly used genes for screening presumptivelytransformed cells include β-glucuronidase (GUS, galactosidase,luciferase and chloramphenicol, acetyltransferase. Jefferson, R.A.,Plant Mol. Biol. Rep. 5:387 (1987), Teeri et al., EMBO J. 8:343 (1989),Koncz et al., Proc. Natl. Acad. Sci U.S.A. 84:131 (1987), DeBlock etal., EMBO J. 3:1681 (1984). Another approach to the identification ofrelatively rare transformation events has been use of a gene thatencodes a dominant constitutive regulator of the Zea mays anthocyaninpigmentation pathway. Ludwig et al., Science 247:449 (1990).

Recently, in vivo methods for visualizing GUS activity that do notrequire destruction of plant tissue have been made available. MolecularProbes publication 2908, Imagene Green™, p. 1-4 (1993) and Naleway etal., J. Cell Biol. 115:151a (1991). However, these in vivo methods forvisualizing GUS activity have not proven useful for recovery oftransformed cells because of low sensitivity, high fluorescentbackgrounds and limitations associated with the use of luciferase genesas selectable markers.

More recently, a gene encoding Green Fluorescent Protein (GFP) has beenutilized as a marker for gene expression in prokaryotic and eukaryoticcells. Chalfie et al., Science 263:802 (1994). GFP and mutants of GFPmay be used as screenable markers.

Promoters—Genes included in expression vectors must be driven bynucleotide sequence comprising a regulatory element, for example, apromoter. Several types of promoters are now well known in thetransformation arts, as are other regulatory elements that can be usedalone or in combination with promoters.

As used herein, “promoter” includes reference to a region of DNAupstream from the start of transcription and involved in recognition andbinding of RNA polymerase and other proteins to initiate transcription.A “plant promoter” is a promoter capable of initiating transcription inplant cells. Examples of promoters under developmental control includepromoters that preferentially initiate transcription in certain tissues,such as leaves, roots, seeds, fibers, xylem vessels, tracheids, orsclerenchyma. Such promoters are referred to as “tissue-preferred”.Promoters which initiate transcription only in certain tissue arereferred to as “tissue-specific”. A “cell type” specific promoterprimarily drives expression in certain cell types in one or more organs,for example, vascular cells in roots or leaves. An “inducible” promoteris a promoter which is under environmental control. Examples ofenvironmental conditions that may effect transcription by induciblepromoters include anaerobic conditions or the presence of light.Tissue-specific, tissue-preferred, cell type specific, and induciblepromoters constitute the class of “non-constitutive” promoters. A“constitutive” promoter is a promoter which is active under mostenvironmental conditions.

A. Inducible Promoters

An inducible promoter is operably linked to a gene for expression incorn. Optionally, the inducible promoter is operably linked to anucleotide sequence encoding a signal sequence which is operably linkedto a gene for expression in corn. With an inducible promoter the rate oftranscription increases in response to an inducing agent.

Any inducible promoter can be used in the instant invention. See Ward etal., Plant Mol. Biol. 22:361-366 (1993). Exemplary inducible promotersinclude, but are not limited to, that from the ACEI system whichresponds to copper (Meft et al., PNAS 90:4567-4571 (1993)); In2 genefrom maize which responds to benzenesulfonamide herbicide safeners(Hershey et al., Mol. Gen Genetics 227:229-237 (1991) and Gatz et al.,Mol. Gen. Genetics 243:32-38 (1994)) or Tet repressor from Tn10 (Gatz etal., Mol. Gen. Genetics 227:229-237 (1991). A particularly preferredinducible promoter is a promoter that responds to an inducing agent towhich plants do not normally respond. An exemplary inducible promoter isthe inducible promoter from a steroid hormone gene, the transcriptionalactivity of which is induced by a glucocorticosteroid hormone. Schena etal., Proc. Natl. Acad. Sci. U.S.A. 88:0421 (1991).

B. Constitutive Promoters

A constitutive promoter is operably linked to a gene for expression incorn or the constitutive promoter is operably linked to a nucleotidesequence encoding a signal sequence which is operably linked to a genefor expression in corn.

Many different constitutive promoters can be utilized in the instantinvention. Exemplary constitutive promoters include, but are not limitedto, the promoters from plant viruses such as the 35S promoter from CaMV(Odell et al., Nature 313:810-812 (1985) and the promoters from suchgenes as rice actin (McElroy et al., Plant Cell 2:163-171 (1990));ubiquitin (Christensen et al., Plant Mol. Biol. 12:619-632 (1989) andChristensen et al., Plant Mol. Bio. 18:675-689 (1992)); pEMU (Last etal., Theor. AppL. Genet. 81:581-588 (1991)); MAS (Velten et al., EMBO J.3:2723-2730 (1984)) and maize H3 histone (Lepetit et al., Mol. Gen.Genetics 231:276-285 (1992) and Atanassova et al., Plant Journal2 (3):291-300 (1992)).

The ALS promoter, Xba1/Ncol fragment 5′ to the Brassica napus ALS3structural gene (or a nucleotide sequence similarity to said Xba1/Ncolfragment), represents a particularly useful constitutive promoter. SeePCT application WO96/30530.

C. Tissue-specific or Tissue-preferred Promoters

A tissue-specific promoter is operably linked to a gene for expressionin corn. Optionally, the tissue-specific promoter is operably linked toa nucleotide sequence encoding a signal sequence which is operablylinked to a gene for expression in corn. Plants transformed with a geneof interest operably linked to a tissue-specific promoter produce theprotein product of the transgene exclusively, or preferentially, in aspecific tissue.

Any tissue-specific or tissue-preferred promoter can be utilized in theinstant invention. Exemplary tissue-specific or tissue-preferredpromoters include, but are not limited to, a root-preferred promoter,such as that from the phaseolin gene (Murai et al., Science 23:476-482(1983) and Sengupta-Gopalan et al., Proc. Natl. Acad. Sci. U.S.A.82:3320-3324 (1985)); a leaf-specific and light-induced promoter such asthat from cab or rubisco (Simpson et al., EMBO J. 4(11):2723-2729 (1985)and Timko et al., Nature 318:579-582 (1985)); an anther-specificpromoter such as that from LAT52 (Twell et al., Mol. Gen. Genetics217:240-245 (1989)); a pollen-specific promoter such as that from Zm13(Guerrero et al., Mol. Gen. Genetics 244:161-168 (1993)) or amicrospore-preferred promoter such as that from apg (Twell et al., Sex.Plant Reprod. 6:217-224 (1993).

Signal Sequences for Targeting Proteins to Subcellular Compartments

Transport of protein produced by transgenes to a subcellular compartmentsuch as the chloroplast, vacuole, peroxisome, glyoxysome, cell wall ormitochondroin or for secretion into the apoplast, is accomplished bymeans of operably linking the nucleotide sequence encoding a signalsequence to the 5′ and/or 3′ region of a gene encoding the protein ofinterest. Targeting sequences at the 5′ and/or 3′ end of the structuralgene may determine, during protein synthesis and processing, where theencoded protein is ultimately compartmentalized.

The presence of a signal sequence directs a polypeptide to either anintracellular organelle or subcellular compartment or for secretion tothe apoplast. Many signal sequences are known in the art. See, forexample Becker et al., Plant Mol. Biol. 20:49 (1992), Close, P.S.,Master's Thesis, Iowa State University (1993), Knox, C., et al.,“Structure and Organization of Two Divergent Alpha-Amylase Genes fromBarley”, Plant Mol. Biol. 9:3-17 (1987), Lerner et al., Plant Physiol.91:124-129 (1989), Fontes et al., Plant Cell 3:483-496 (1991), Matsuokaet al., Proc. Natl. Acad. Sci. 88:834 (1991), Gould et al., J Cell.Biol. 108:1657 (1989), Creissen et al., Plant J.2:129 (1991), Kalderon,et al., A short amino acid sequence able to specify nuclear location,Cell 39:499-509 (1984), Steifel, et al., Expression of a maize cell wallhydroxyproline-rich glycoprotein gene in early leaf and root vasculardifferentiation, Plant Cell 2:785-793 (1990).

Foreign Protein Genes and Agronomic Genes

With transgenic plants according to the present invention, a foreignprotein can be produced in commercial quantities. Thus, techniques forthe selection and propagation of transformed plants, which are wellunderstood in the art, yield a plurality of transgenic plants which areharvested in a conventional manner, and a foreign protein then can beextracted from a tissue of interest or from total biomass. Proteinextraction from plant biomass can be accomplished by known methods whichare discussed, for example, by Heney and Orr, Anal. Biochem. 114:92-6(1981).

According to a preferred embodiment, the transgenic plant provided forcommercial production of foreign protein is corn. In another preferredembodiment, the biomass of interest is seed. For the relatively smallnumber of transgenic plants that show higher levels of expression, agenetic map can be generated, primarily via conventional RFLP, PCR andSSR analysis, which identifies the approximate chromosomal location ofthe integrated DNA molecule. For exemplary methodologies in this regard,see Glick and Thompson, Methods in Plant Molecular Biology andBiotechnology CRC Press, Boca Raton 269:284 (1993). Map informationconcerning chromosomal location is useful for proprietary protection ofa subject transgenic plant. If unauthorized propagation is undertakenand crosses made with other germplasm, the map of the integration regioncan be compared to similar maps for suspect plants, to determine if thelatter have a common parentage with the subject plant. Map comparisonswould involve hybridizations, RFLP, PCR, SSR and sequencing, all ofwhich are conventional techniques.

Likewise, by means of the present invention, agronomic genes can beexpressed in transformed plants. More particularly, plants can begenetically engineered to express various phenotypes of agronomicinterest. Exemplary genes implicated in this regard include, but are notlimited to, those categorized below:

1. Genes That Confer Resistance to Pests or Disease and That Encode:

A. Plant disease resistance genes. Plant defenses are often activated byspecific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant inbred line can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example Jones et al., Science 266:789 (1994)(cloning of the tomato Cf-9 gene for resistance to Cladosporium fulvum);Martin et al., Science 262:1432 (1993) (tomato Pto gene for resistanceto Pseudomonas syringae pv. Tomato encodes a protein kinase); Mindrinoset al., Cell 78:1089 (1994) (Arabidopsis RSP2 gene for resistance toPseudomonas syringae).

B. A Bacillus thuringiensis protein, a derivative thereof or a syntheticpolypeptide modeled thereon. See, for example, Geiser et al., Gene48:109 (1986), who disclose the cloning and nucleotide sequence of a Btδ-endotoxin gene. Moreover, DNA molecules encoding δ-endotoxin genes canbe purchased from American Type Culture Collection, Manassas, Va., forexample, under ATCC Accession Nos. 40098, 67136, 31995 and 31998.

C. A lectin. See, for example, the disclose by Van Damme et al., PlantMolec. Biol. 24:25 (1994), who disclose the nucleotide sequences ofseveral Clivia miniata man nose-binding lectin genes.

D. A vitamin-binding protein such as avidin. See PCT applicationUS93/06487, the contents of which are hereby incorporated by reference.The application teaches the use of avidin and avidin homologues aslarvicides against insect pests.

E. An enzyme inhibitor, for example, a protease or proteinase inhibitoror an amylase inhibitor. See, for example, Abe et al., J. Biol. Chem.262:16793 (1987) (nucleotide sequence of rice cysteine proteinaseinhibitor), Huub et al., Plant Molec. Biol. 21:985 (1993) (nucleotidesequence of cDNA encoding tobacco proteinase inhibitor 1), Sumitani etal., Biosci. Biotech. Biochem. 57:1243 (1993) (nucleotide sequence ofStreptomyces nitrosporeus α-amylase inhibitor).

F. An insect-specific hormone or pheromone such as an ecdysteroid andjuvenile hormone, a variant thereof, a mimetic based thereon, or anantagonist or agonist thereof. See, for example, the disclosure byHammock et al., Nature 344:458 (1990), of baculovirus expression ofcloned juvenile hormone esterase, an inactivator of juvenile hormone.

G. An insect-specific peptide or neuropeptide which, upon expression,disrupts the physiology of the affected pest. For example, see thedisclosures of Regan, J. Biol. Chem. 269:9 (1994) (expression cloningyields DNA coding for insect diuretic hormone receptor), and Pratt etal., Biochem. Biophys. Res. Comm. 163:1243 (1989) (an allostatin isidentified in Diploptera puntata). See also U.S. Pat. No. 5,266,317 toTomalski et al., who disclose genes encoding insect-specific, paralyticneurotoxins.

H. An insect-specific venom produced in nature by a snake, a wasp, etc.For example, see Pang et al., Gene 116:165 (1992), for disclosure ofheterologous expression in plants of a gene coding for a scorpioninsectotoxic peptide.

I. An enzyme responsible for a hyper accumulation of a monterpene, asesquiterpene, a steroid, hydroxamic acid, a phenylpropanoid derivativeor another non-protein molecule with insecticidal activity.

J. An enzyme involved in the modification, including thepost-translational modification, of a biologically active molecule; forexample, a glycolytic enzyme, a proteolytic enzyme, a lipolytic enzyme,a nuclease, a cyclase, a transaminase, an esterase, a hydrolase, aphosphatase, a kinase, a phosphorylase, a polymerase, an elastase, achitinase and a glucanase, whether natural or synthetic. See PCTapplication WO 93/02197 in the name of Scott et al., which discloses thenucleotide sequence of a callase gene. DNA molecules which containchitinase-encoding sequences can be obtained, for example, from the ATCCunder Accession Nos. 39637 and 67152. See also Kramer et al., InsectBiochem. Molec. Biol. 23:691 (1993), who teach the nucleotide sequenceof a cDNA encoding tobacco hookworm chitinase, and Kawalleck et al.,Plant Molec. Biol. 21:673 (1993), who provide the nucleotide sequence ofthe parsley ubi4-2 polyubiquitin gene.

K. A molecule that stimulates signal transduction. For example, see thedisclosure by Botella et al., Plant Molec. Biol. 24:757 (1994), ofnucleotide sequences for mung bean calmodulin cDNA clones, and Griess etal., Plant Physiol. 104:1467 (1994), who provide the nucleotide sequenceof a maize calmodulin cDNA clone.

L. A hydrophobic moment peptide. See PCT application WO95/16776(disclosure of peptide derivatives of Tachyplesin which inhibit fungalplant pathogens) and PCT application WO95/18855 (teaches syntheticantimicrobial peptides that confer disease resistance), the respectivecontents of which are hereby incorporated by reference.

M. A membrane permease, a channel former or a channel blocker. Forexample, see the disclosure of Jaynes et al., Plant Sci 89:43 (1993), ofheterologous expression of a cecropin-β, lytic peptide analog to rendertransgenic tobacco plants resistant to Pseudomonas solanacearum.

N. A viral-invasive protein or a complex toxin derived therefrom. Forexample, the accumulation of viral coat proteins in transformed plantcells imparts resistance to viral infection and/or disease developmenteffected by the virus from which the coat protein gene is derived, aswell as by related viruses. See Beachy et al., Ann. rev. Phytopathol.28:451 (1990). Coat protein-mediated resistance has been conferred upontransformed plants against alfalfa mosaic virus, cucumber mosaic virus,tobacco streak virus, potato virus X, potato virus Y, tobacco etchvirus, tobacco rattle virus and tobacco mosaic virus. Id.

O. An insect-specific antibody or an immunotoxin derived therefrom.Thus, an antibody targeted to a critical metabolic function in theinsect gut would inactivate an affected enzyme, killing the insect. Cf.Taylor et al., Abstract #497, Seventh Int'l Symposium on MolecularPlant-Microbe Interactions (Edinburgh, Scotland) (1994) (enzymaticinactivation in transgenic tobacco via production of single-chainantibody fragments).

P. A virus-specific antibody. See, for example, Tavladoraki et al.,Nature 366:469 (1993), who show that transgenic plants expressingrecombinant antibody genes are protected from virus attack.

Q. A developmental-arrestive protein produced in nature by a pathogen ora parasite. Thus, fungal endo α-1, 4-D-polygalacturonases facilitatefungal colonization and plant nutrient release by solubilizing plantcell wall homo-α-1,4-D-galacturonase. See Lamb et al., Bio/Technology10:1436 (1992). The cloning and characterization of a gene which encodesa bean endopolygalacturonase-inhibiting protein is described by Toubartet al., Plant J. 2:367 (1992).

R. A development-arrestive protein produced in nature by a plant. Forexample, Logemann et al., Bioi/Technology 10:305 (1992), have shown thattransgenic plants expressing the barley ribosome-inactivating gene havean increased resistance to fungal disease.

2. Genes That Confer Resistance to a Herbicide, For Example:

A. A herbicide that inhibits the growing point or meristem, such as animidazalinone or a sulfonylurea. Exemplary genes in this category codefor mutant ALS and AHAS enzyme as described, for example, by Lee et al.,EMBO J. 7:1241 (1988), and Miki et al., Theor. AppL Genet. 80:449(1990), respectively.

B. Glyphosate (resistance impaired by mutant5-enolpyruvl-3-phosphikimate synthase (EPSP) and aroA genes,respectively) and other phosphono compounds such as glufosinate(phosphinothricin acetyl transferase, PAT and Streptomyces hygroscopicusphosphinothricin-acetyl transferase, bar, genes), and pyridinoxy orphenoxy propionic acids and cycloshexones (ACCase inhibitor-encodinggenes). See, for example, U.S. Pat. No. 4,940,835 to Shah, et al., whichdiscloses the nucleotide sequence of a form of EPSP which can conferglyphosate resistance. A DNA molecule encoding a mutant aroA gene can beobtained under ATCC accession number 39256, and the nucleotide sequenceof the mutant gene is disclosed in U.S. Pat. No. 4,769,061 to Comai.European patent application No. 0 333 033 to Kumada et al., and U.S.Pat. No. 4,975,374 to Goodman et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in Europeanapplication No. 0 242 246 to Leemans et al., DeGreef et al.,Bio/Technology 7:61 (1989), describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. Exemplary of genes conferring resistance tophenoxy propionic acids and cycloshexones, such as sethoxydim andhaloxyfop are the Acc1-S1, Accl-S2 and Acc1-S3 genes described byMarshall et al., Theor. Appl. Genet. 83:435 (1992).

C. A herbicide that inhibits photosynthesis, such as a triazine (psbAand gs+genes) and a benzonitrile (nitrilase gene). Przibilla et al.,Plant Cell 3:169 (1991), describe the transformation of Chlamydomonaswith plasmids encoding mutant psbA genes. Nucleotide sequences fornitrilase genes are disclosed in U.S. Pat. No. 4,810,648 to Stalker, andDNA molecules containing these genes are available under ATCC AccessionNos. 53435, 67441, and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes et al., Biochem. J.285:173 (1992).

3. Genes That Confer or Contribute to a Value-Added Trait, Such as:

A. Modified fatty acid metabolism, for example, by transforming a plantwith an antisense gene of stearyl-ACP desaturase to increase stearicacid content of the plant. See Knultzon et al., Proc. Natl. Acad. Sci.U.S.A. 89:2624 (1992).

B. Decreased phytate content, 1) Introduction of a phytase-encoding genewould enhance breakdown of phytate, adding more free phosphate to thetransformed plant. For example, see Van Hartingsveldt et al., Gene127:87 (1993), for a disclosure of the nucleotide sequence of anAspergillus niger phytase gene.

2) A gene could be introduced that reduced phytate content. In maize,this, for example, could be accomplished, by cloning and thenreintroducing DNA associated with the single allele which is responsiblefor maize mutants characterized by low levels of phytic acid. See Raboyet al., Maydica 35:383 (1990).

C. Modified carbohydrate composition effected, for example, bytransforming plants with a gene coding for an enzyme that alters thebranching pattern of starch. See Shiroza et al., J. Bacteol. 170:810(1988) (nucleotide sequence of Streptococcus mutantsfructosyltransferase gene), Steinmetz et al., Mol. Gen. Genet. 20:220(1985) (nucleotide sequence of Bacillus subtilis levansucrase gene), Penet al., Bio/Technology 10:292 (1992) (production of transgenic plantsthat express Bacillus lichenifonnis α-amylase), Elliot et al., PlantMolec. Biol. 21:515 (1993) (nucleotide sequences of tomato invertasegenes), Søgaard et al., J. Biol. Chem. 268:22480 (1993) (site-directedmutagenesis of barley α-amylase gene), and Fisher et al., Plant Physiol.102:1045 (1993) (maize endosperm starch branching enzyme II).

Methods for Corn Transformation

Numerous methods for plant transformation have been developed, includingbiological and physical, plant transformation protocols. See, forexample, Miki et al., “Procedures for Introducing Foreign DNA intoPlants” in Methods in Plant Molecular Biology and Biotechnology, GlickB. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993) pages67-88. In addition, expression vectors and in vitro culture methods forplant cell or tissue transformation and regeneration of plants areavailable. See, for example, Gruber et al., “Vectors for PlantTransformation” in Methods in Plant Molecular Biology and Biotechnology,Glick B. R. and Thompson, J. E. Eds. (CRC Press, Inc., Boca Raton, 1993)pages 89-119.

A. Agrobacterium-mediated Transformation

One method for introducing an expression vector into plants is based onthe natural transformation system of Agrobacterium. See, for example,Horsch et al., Science 227:1229 (1985). A. tumefaciens and A. rhizogenesare plant pathogenic soil bacteria which genetically transform plantcells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes,respectively, carry genes responsible for genetic transformation of theplant. See, for example, Kado, C. I., Crit. Rev. Plant Sci. 10:1 (1991).Descriptions of Agrobacterium vector systems and methods forAgrobacterium-mediated gene transfer are provided by Gruber et al.,supra, Miki et al., supra, and Moloney et al., Plant Cell Reports 8:238(1989). See also, U.S. Pat. No. 5,591,616 issued Jan. 7, 1997.

B. Direct Gene Transfer

Despite the fact the host range for Agrobacterium-mediatedtransformation is broad, some major cereal crop species and gymnospermshave generally been recalcitrant to this mode of gene transfer, eventhough some success has recently been achieved in rice and corn. Hiei etal., The Plant Journal 6:271-282 (1994) and U.S. Pat. No. 5,591,616issued Jan. 7, 1997. Several methods of plant transformation,collectively referred to as direct gene transfer, have been developed asan alternative to Agrobacterium-mediated transformation.

A generally applicable method of plant transformation ismicroprojectile-mediated transformation wherein DNA is carried on thesurface of microprojectiles measuring 1 to 4 μm. The expression vectoris introduced into plant tissues with a biolistic device thataccelerates the microprojectiles to speeds of 300 to 600 m/s which issufficient to penetrate plant cell walls and membranes. Sanford et al.,Part. Sci. Technol. 5:27 (1987), Sanford, J.C., Trends Biotech. 6:299(1988), Klein et al., Bio/Technology 6:559-563 (1988), Sanford, J.C.,Physiol Plant 7:206 (1990), Klein et al., Biotechnology 10:268 (1992).In corn, several target tissues can be bombarded with DNA-coatedmicroprojectiles in order to produce transgenic plants, including, forexample, callus (Type I or Type II), immature embryos, and meristematictissue.

Another method for physical delivery of DNA to plants is sonication oftarget cells. Zhang et al., Bio/Technology 9:996 (1991). Alternatively,liposome or spheroplast fusion have been used to introduce expressionvectors into plants. Deshayes et al., EMBO J., 4:2731 (1985), Christouet al., Proc Natl. Acad. Sci. U.S.A. 84:3962 (1987). Direct uptake ofDNA into protoplasts using CaCI₂ precipitation, polyvinyl alcohol orpoly-L-omithine have also been reported. Hain et al., Mol. Gen. Genet.199:161 (1985) and Draper et al., Plant Cell Physiol. 23:451 (1982).Electroporation of protoplasts and whole cells and tissues have alsobeen described. Donn et al., In Abstracts of Vllth InternationalCongress on Plant Cell and Tissue Culture IAPTC, A2-38, p 53 (1990);D'Halluin et al., Plant Cell4:1495-1505 (1992) and Spencer et al., PlantMol. Biol. 24:51-61 (1994).

Following transformation of corn target tissues, expression of theabove-described selectable marker genes allows for preferentialselection of transformed cells, tissues and/or plants, usingregeneration and selection methods now well known in the art.

The foregoing methods for transformation would typically be used forproducing a transgenic inbred line. The transgenic inbred line couldthen be crossed, with another (non-transformed or transformed) inbredline, in order to produce a new transgenic inbred line. Alternatively, agenetic trait which has been engineered into a particular corn lineusing the foregoing transformation techniques could be moved intoanother line using traditional backcrossing techniques that are wellknown in the plant breeding arts. For example, a backcrossing approachcould be used to move an engineered trait from a public, non-eliteinbred line into an elite inbred line, or from an inbred line containinga foreign gene in its genome into an inbred line or lines which do notcontain that gene. As used herein, “crossing” can refer to a simple X byY cross, or the process of backcrossing, depending on the context.

When the term inbred corn plant is used in the context of the presentinvention, this also includes any single gene conversions of thatinbred. The term single gene converted plant as used herein refers tothose corn plants which are developed by a plant breeding techniquecalled backcrossing wherein essentially all of the desired morphologicaland physiological characteristics of an inbred are recovered in additionto the single gene transferred into the inbred via the backcrossingtechnique. Backcrossing methods can be used with the present inventionto improve or introduce a characteristic into the inbred. The termbackcrossing as used herein refers to the repeated crossing of a hybridprogeny back to one of the parental corn plants for that inbred. Theparental corn plant which contributes the gene for the desiredcharacteristic is termed the nonrecurrent or donor parent. Thisterminology refers to the fact that the nonrecurrent parent is used onetime in the backcross protocol and therefore does not recur. Theparental corn plant to which the gene or genes from the nonrecurrentparent are transferred is known as the recurrent parent as it is usedfor several rounds in the backcrossing protocol (Poehlman & Sleper,1994; Fehr, 1987). In a typical backcross protocol, the original inbredof interest (recurrent parent) is crossed to a second inbred(nonrecurrent parent) that carries the single gene of interest to betransferred. The resulting progeny from this cross are then crossedagain to the recurrent parent and the process is repeated until a cornplant is obtained wherein essentially all of the desired morphologicaland physiological characteristics of the recurrent parent are recoveredin the converted plant, in addition to the single transferred gene fromthe nonrecurrent parent.

The selection of a suitable recurrent parent is an important step for asuccessful backcrossing procedure. The goal of a backcross protocol isto alter or substitute a single trait or characteristic in the originalinbred. To accomplish this, a single gene of the recurrent inbred ismodified or substituted with the desired gene from the nonrecurrentparent, while retaining essentially all of the rest of the desiredgenetic, and therefore the desired physiological and morphological,constitution of the original inbred. The choice of the particularnonrecurrent parent will depend on the purpose of the backcross, one ofthe major purposes is to add some commercially desirable, agronomicallyimportant trait to the plant. The exact backcrossing protocol willdepend on the characteristic or trait being altered to determine anappropriate testing protocol. Although backcrossing methods aresimplified when the characteristic being transferred is a dominantallele, a recessive allele may also be transferred. In this instance itmay be necessary to introduce a test of the progeny to determine if thedesired characteristic has been successfully transferred.

Many single gene traits have been identified that are not regularlyselected for in the development of a new inbred but that can be improvedby backcrossing techniques. Single gene traits may or may not betransgenic, examples of these traits include but are not limited to,male sterility, waxy starch, herbicide resistance, resistance forbacterial, fungal, or viral disease, insect resistance, male fertility,enhanced nutritional quality, industrial usage, yield stability andyield enhancement. These genes are generally inherited through thenucleus. Some known exceptions to this are the genes for male sterility,some of which are inherited cytoplasmically, but still act as singlegene traits. Several of these single gene traits are described in U.S.Pat. Nos. 5,777,196; 5,948,957 and 5,969,212, the disclosures of whichare specifically hereby incorporated by reference.

Industrial Applicability

Corn is used as human food, livestock feed, and as raw material inindustry. The food uses of corn, in addition to human consumption ofcorn kernels, include both products of dry- and wet-milling industries.The principal products of corn dry milling are grits, meal and flour.The corn wet-milling industry can provide corn starch, corn syrups, anddextrose for food use. Corn oil is recovered from corn germ, which is aby-product of both dry- and wet-milling industries.

Corn, including both grain and non-grain portions of the plant, is alsoused extensively as livestock feed, primarily for beef cattle, dairycattle, hogs and poultry.

Industrial uses of corn include production of ethanol, corn starch inthe wet-milling industry and corn flour in the dry-milling industry. Theindustrial applications of corn starch and flour are based on functionalproperties, such as viscosity, film formation, adhesive properties, andability to suspend particles. The corn starch and flour have applicationin the paper and textile industries. Other industrial uses includeapplications in adhesives, building materials, foundry binders, laundrystarches, explosives, oil-well muds and other mining applications.

Plant parts other than the grain of corn are also used in industry, forexample: stalks and husks are made into paper and wallboard and cobs areused for fuel and to make charcoal.

The seed of inbred corn line 7RN401, the plant produced from the inbredseed, the hybrid corn plant produced from the crossing of the inbred,hybrid seed, and various parts of the hybrid corn plant and transgenicversions of the foregoing, can be utilized for human food, livestockfeed, and as a raw material in industry.

TABLES

In Table 1 that follows the average inbred by tester performance isshown comparing 7RN401 to LH132 crossed to the same testers and grown inthe same environments.

In Table 2 that follows the average inbred by tester performance isshown comparing 7RN401 x Tester A to Pioneer 33A14 grown in the sameenvironments.

TABLE 1 7RN401 × LH132 × Traits Tester A Tester A Replications Yield 196184 296 Moisture 19.5 19.1 296 CTPS % 112.3 106.4 Stalk Lodging % 0.80.8 269 Root Lodging % 1.2 0.5 265 Dropped Ears 5 4 168 Top Integrity7.6 7.8 170 Plant Height 91 91 67 Ear Height 43 40 67 Final Population29.2 29.3 296 Adjusted Test Weight 57.2 57.8 189 GDDS 1323 1300 7 b 0.90.9 Environments 108 108 Years 2 2 2 Emergence Vigor 5.9 5.6 8 Oil % 4.24.1 3 Protein % 10.5 11.4 3 Starch % 71.1 70.1 3 Gray Leaf Spot 5.1 4.58 North. Corn Leaf Blight 5.9 5.9 8 South. Corn Leaf Blight 7.4 7.3 4Eyespot 7.1 7.6 6 Stewart's Wilt Leaf Blight 6.2 6.8 4 Fusarium KernelRot 5.2 5.0 4

TABLE 2 7RN401 × Traits Tester A Pioneer 33A14 Replications Yield 197190 273 Moisture 19.4 18.9 273 CTPS % 112.4 108.3 Stalk Lodging % 0.81.7 246 Root Lodging % 0.8 0.5 242 Dropped Ears 5 1 158 Top Integrity7.7 6.8 160 Plant Height 91 90 65 Ear Height 43 44 65 Final Population29.2 29.3 273 Adjusted Test Weight 57.1 58.7 174 GDDS 1380 1407 6 b 0.90.9 Environments 104 104 Years 2 2 Emergence Vigor 5.9 5.9 8 Oil % 4.33.2 2 Protein % 10.2 10.5 2 Starch % 71.7 73 2 Gray Leaf Spot 5.1 4.8 8North. Corn Leaf Blight 5.9 6.0 8 South. Corn Leaf Blight 7.4 6.8 4Eyespot 7.1 6.7 6 Stewart's Wilt Leaf Blight 6.2 6.8 4 Fusarium KernelRot 5.2 5.8 4

In Table 4 listed below the kernel grade data for the instant inventionand other inbreds of similar maturity are given. This hand screeningprocedure to determine the percent for each kernel size is performed asfollows:

1. Weigh out a 500 gram working sample and shake for 2.5 minutes on ahand-screen shaker using the 24/64-, 21/64-, 19/64-, 17/64- and15/64-inch round-hole screens.

2. Place seed remaining on the 24/64- or falling through the 15/64-inchscreens in the discard fraction.

3. Separate rounds from flats by shaking for 2.5 minutes using the13.5/64-, 13/64-, 12.5/64- and 12/64-inch slotted screens as indicatedbelow.

4. Remove and discard any foreign material such as cob tips, shanks,etc., insect or disease damaged kernels and seeds that are less than 1/2of a whole kernel.

5. Weigh the clean seeds in grams to determine gradeout percentages byweight.

TABLE 3 Seed Size Seed Size Over Slotted Through Slotted FractionSlotted Screen Screen Screen Over 24/64 Discard — — Over 21/64 13½/64 LRLF Over 19/64 13/64 MR1 MF1 Over 17/64 12½/64 MR2 MF2 Over 15/64 12/64SR SF Through 15/64 Discard — —

TABLE 4 Percentage of Kernel Sizes Dis- SR SF MR2 MF2 MR1 MF1 LR LF cardInbred Seeds/lb % % % % % % % % % 7RN401 1656 6.1 4.7 21.6 21.1 20.315.0 6.1 3.2 1.9 LH132 1654 5.3 4.2 21.1 21.2 19.7 19.1 4.0 2.9 2.6LH198 1602 7.3 3.8 20.8 20.2 20.3 18.9 3.8 3.3 1.7

Deposit Information

A deposit of the Agrigenetics, Inc. d/b/a Mycogen Seeds prporietaryinbred corn line 7RN401 disclosed above and recited in the appendedclaims has been made with the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 201101. The date of depositwas May 29, 2002. The deposit of 2,500 seeds were taken from the samedeposit maintained by Agrigenetics, Inc. d/b/a Mycogen Seeds since priorto the filing date of this application. All restrictions upon thedeposit have been removed, and the deposit is intended to meet all ofthe requirements of 37 C.F.R. §1801-1809. The ATCC accession no. isPTA-4415. The deposit will be maintained in the depository for a periodof 30 years, or 5 years after the last request, or for the effectivelife of the patent, whichever is longer, and will be replaced asnecessary during that period.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity andunderstanding. However, it will be obvious that certain changes andmodifications such as single gene modifications and mutations,somoclonal variants, variant individuals selected from large populationsof the plants of the instant inbred and the like may be practiced withinthe scope of the invention, as limited only by the scope of the appendedclaims.

What is claimed is:
 1. Seed of corn inbred line designated 7RN401,representative seed of said line having been deposited under ATCCAccession No. PTA-4415.
 2. A corn plant, or parts thereof, produced bygrowing the seed of claim
 1. 3. Pollen of the plant of claim
 2. 4. Anovule of the plant of claim
 2. 5. The corn plant of claim 2, furthercomprising a genetic factor conferring male sterility.
 6. A corn plantwith all of the physiological and morphological characteristics of corninbred 7RN401, a sample of said seed having been deposited under ATCCAccession No. PTA-4415, wherein said corn plant is produced by a tissueculture process using the corn plant of claim 2 as the starting materialfor such a process.
 7. A method for producing a hybrid corn seedcomprising crossing a first inbred parent corn plant with a secondinbred parent corn plant and harvesting the resultant hybrid corn seed,wherein said first inbred parent corn plant or second said parent cornplant is the corn plant of claim
 2. 8. A hybrid corn seed produced bythe method of claim
 11. 9. A hybrid corn plant, or parts thereof,produced by growing said hybrid corn seed of claim
 8. 10. A corn seedproduced by growing said corn plant of claim 9 and harvesting theresultant corn seed.
 11. An F₁ hybrid seed produced by crossing theinbred corn plant according to claim 2 with another, different cornplant.